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1.
Bioconjug Chem ; 27(3): 824-30, 2016 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-26886721

RESUMEN

We have developed a novel pH-sensitive linker based on a phosphoramidate scaffold that can be tuned to release amine-containing drug molecules at various pH values. The pH-triggered phosphoramidate-based linkers are responsive to pH alone and do not require intracellular enzymatic action to initiate drug release. Key to the pH-triggered amine release from these linkers is a proximal acidic group (e.g., pyridinium or carboxylic acid) to promote the hydrolysis of the phosphoramidate P-N bond, presumably through an intramolecular general-acid type mechanism. Phosphoramidate hydrolysis is largely governed by the pKa of the leaving amine (e.g., primary, secondary, aniline). However, the proximity of the neighboring pyridine group attenuates the stability of the P-N bond to hydrolysis, thus allowing for control over the release of an amine from the phosphoramidate center. Based on the model scaffolds examined, phosphoramidate-based linkers could be selected for particular properties for controlled-release applications such as amine type, stability under physiological conditions, or release rates at various pH values such as intracellular endosomal conditions. The tunability of the phosphoramidate scaffold is expected to find broad applicability in various controlled drug-release applications such as antibody or small-molecule drug conjugates, drug-eluting stents, prodrug activation, as well as intracellular trafficking studies in which pH changes can trigger the release of turn-on dyes.


Asunto(s)
Preparaciones de Acción Retardada/química , Concentración de Iones de Hidrógeno , Antineoplásicos/administración & dosificación
2.
Bioconjug Chem ; 27(9): 2206-13, 2016 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-27562353

RESUMEN

We developed a second generation of tunable pH-sensitive linkers based on our phosphoramidate scaffold to release amine-containing drugs under acidic conditions. The pH-triggered phosphoramidate-based linkers are responsive to pH and do not require intracellular enzymatic action to initiate drug release. On the basis of the model scaffolds examined, phosphoramidate-based linkers were selected for particular properties for controlled release applications such as amine type, stability under physiological conditions, or release rates at various pH values such as intracellular endosomal conditions. Key to the pH-triggered amine release from these linker is a proximal carboxylic acid to promote hydrolysis of the phosphoramidate P-N bond, presumably through an intramolecular general acid-type mechanism. Phosphoramidate hydrolysis is largely governed by the pKa of the leaving amine. However, the proximity of the neighboring carboxylic acid attenuates the stability of the P-N bond to hydrolysis, thus allowing for control over the release of an amine from the phosphoramidate center. In addition, we observed that the Thorpe-Ingold effect and rigidification of the scaffold could further enhance the rate of release. Esterification of the neighboring carboxylic acid was found to protect the scaffold from rapid release at low pH. This latter observation is particularly noteworthy as it suggests that the phosphoramidate-based drug-conjugate scaffold can be protected as an ester prodrug for oral administration. While the tunability phosphoramidate linkers is attractive for applications in intracellular trafficking studies in which pH changes can trigger release of turn-on dyes, antibody drug conjugates, small-molecule drug conjugates, and drug eluting stents (DES), the promise of oral delivery of drug conjugates is expected to have broad impact in controlled release applications.


Asunto(s)
Amidas/química , Portadores de Fármacos/química , Ácidos Fosfóricos/química , Preparaciones de Acción Retardada , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Hidrólisis , Temperatura
3.
J Org Chem ; 80(17): 8705-12, 2015 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-26270193

RESUMEN

Proton-coupled electron transfer (PCET) model systems combine one-electron oxidants and bases to generate net hydrogen atom acceptors. We have generated two persistent pyridyl-appended radical cations: 10-(pyrid-2-yl)-10H-phenothiazinium (PPT•+) and 3-(pyrid-2-yl)-10-methyl-10H-phenothiazinium (MPTP•+). EPR spectra and corresponding calculations indicate phenothiazinium radical cations with minimal spin on the pyridine nitrogen. Addition of hindered phenols causes the radical cations to decay, and protonated products and the corresponding phenoxyl radicals to form. The ΔG° values for the formation of intermediates (determined through cyclic voltammetry and pKa measurements) rule out a stepwise mechanism, and kinetic isotope effects support concerted proton­electron transfer (CPET) as the mechanism. Calculations indicate that the reaction of PPT•+ + tBu3PhOH undergoes a significant conformational change with steric interactions on the diabatic surface while maintaining the hydrogen bond; in contrast, MPTP•+ + tBu3PhOH maintains its conformation throughout the reaction. This difference is reflected in both experiment and calculations with ΔG(⧧)MPTP•+ < ΔG(⧧)PPT•+ despite ΔG°MPTP•+ > ΔG°PPT•+. Experimental results with 2,6-di-tert-butyl-4-methoxyphenol are similar. Hence, despite the structural similarity between the compounds, differences in the inner sphere component for CPET affect the kinetics.

4.
Prostate ; 74(5): 451-7, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24449207

RESUMEN

BACKGROUND: Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. METHODS: Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. RESULTS: Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1) mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50 = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. CONCLUSIONS: The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans.


Asunto(s)
Adenocarcinoma/metabolismo , Antígenos de Superficie/metabolismo , Glutamato Carboxipeptidasa II/metabolismo , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Perros , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Masculino , Próstata/efectos de los fármacos , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/patología
5.
Mol Imaging Biol ; 22(2): 274-284, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31321650

RESUMEN

PURPOSE: Prostate-specific membrane antigen (PSMA) continues to be the hallmark biomarker for prostate cancer as it is expressed on nearly all prostatic tumors. In addition, increased PSMA expression correlates with castration resistance and progression to the metastatic stage of the disease. Recently, we combined both an albumin-binding motif and an irreversible PSMA inhibitor to develop the novel PSMA-targeted radiotherapeutic agent, CTT1403. This molecule was novel in the field of PSMA-targeted agents as its key motifs resulted in extended blood circulation time and tumor uptake, rapid and extensive internalization into PSMA+ cells, and promising therapeutic efficacy. The objective of this study was to perform IND-enabling translational studies on CTT1403 in rodent models. PROCEDURES: A dose optimization study was performed in PC3-PIP (PSMA+) tumor-bearing mice. Treatment groups were randomly selected to receive one to three 14-MBq injections of CTT1403. Control groups included (1) saline, (2) non-radioactive [175Lu]CTT1403, or (3) two injections of 14 MBq CTT1751, a Lu-177-labeled non-targeted albumin-binding moiety. Tumor growth was monitored up to 120 days. Small-animal single photon emission tomography/X-ray computed tomography imaging was performed with CTT1403 and CTT1751 in PC3-PIP tumor-bearing mice to visualize infiltration of the Lu-177-labeled agent into the tumor. In preparation for a first-in-human study, human absorbed doses were estimated based on rat biodistribution out to 5 weeks to determine a safe CTT1403 therapy dose in humans. RESULTS: Two to 3 injections of 14 MBq CTT1403 yielded significant tumor growth inhibition and increased survival compared with all control groups and mice receiving 1 injection of 14 MBq CTT1403. Five of 12 mice receiving 2 or 3 injections of CTT1403 survived to the 120-day post-treatment study endpoint. Dosimetry identified the kidneys as the dose-limiting organ, with an equivalent dose of 5.18 mSv/MBq, resulting in a planned maximum dose of 4.4 GBq for phase 1 clinical trials. CONCLUSIONS: The preclinical efficacy and dosimetry of CTT1403 suggest that this agent has significant potential to be safe and effective in humans.


Asunto(s)
Lutecio/farmacología , Radioisótopos/farmacología , Radiometría/métodos , Radiofármacos/farmacología , Animales , Antígenos de Superficie/química , Ensayos de Selección de Medicamentos Antitumorales , Glutamato Carboxipeptidasa II/química , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Radioisótopos/química , Ratas , Ratas Sprague-Dawley , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Distribución Tisular , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Theranostics ; 7(7): 1928-1939, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28638478

RESUMEN

Prostate-specific membrane antigen (PSMA) continues to be an active biomarker for small-molecule PSMA-targeted imaging and therapeutic agents for prostate cancer and various non-prostatic tumors that are characterized by PSMA expression on their neovasculature. One of the challenges for small-molecule PSMA inhibitors with respect to delivering therapeutic payloads is their rapid renal clearance. In order to overcome this pharmacokinetic challenge, we outfitted a 177Lu-labeled phosphoramidate-based PSMA inhibitor (CTT1298) with an albumin-binding motif (CTT1403) and compared its in vivo performance with that of an analogous compound lacking the albumin-binding motif (CTT1401). The radiolabeling of CTT1401 and CTT1403 was achieved using click chemistry to connect 177Lu-DOTA-N3 to the dibenzocyclooctyne (DBCO)-bearing CTT1298 inhibitor cores. A direct comparison in vitro and in vivo performance was made for CTT1401 and CTT1403; the specificity and efficacy by means of cellular uptake and internalization, biodistribution, and therapeutic efficacy were determined for both compounds. While both compounds displayed excellent uptake and rapid internalization in PSMA+ PC3-PIP cells, the albumin binding moiety in CTT1403 conferred clear advantages to the PSMA-inhibitor scaffold including increased circulating half-life and prostate tumor uptake that continued to increase up to 168 h post-injection. This increased tumor uptake translated into superior therapeutic efficacy of CTT1403 in PSMA+ PC3-PIP human xenograft tumors.


Asunto(s)
Amidas/farmacología , Antineoplásicos/farmacología , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Lutecio/farmacología , Ácidos Fosfóricos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Radioisótopos/farmacología , Albúminas/metabolismo , Amidas/administración & dosificación , Amidas/farmacocinética , Animales , Antígenos de Superficie , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Lutecio/administración & dosificación , Lutecio/farmacocinética , Masculino , Ratones , Ratones Desnudos , Ácidos Fosfóricos/administración & dosificación , Ácidos Fosfóricos/farmacocinética , Radioisótopos/administración & dosificación , Radioisótopos/farmacocinética , Resultado del Tratamiento
7.
J Med Chem ; 59(12): 5684-94, 2016 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-27228467

RESUMEN

A series of phosphoramidate-based prostate specific membrane antigen (PSMA) inhibitors of increasing lipophilicity were synthesized (4, 5, and 6), and their fluorine-18 analogs were evaluated for use as positron emission tomography (PET) imaging agents for prostate cancer. To gain insight into their modes of binding, they were also cocrystallized with the extracellular domain of PSMA. All analogs exhibited irreversible binding to PSMA with IC50 values ranging from 0.4 to 1.3 nM. In vitro assays showed binding and rapid internalization (80-95%, 2 h) of the radiolabeled ligands in PSMA(+) cells. In vivo distribution demonstrated significant uptake in CWR22Rv1 (PSMA(+)) tumor, with tumor to blood ratios of 25.6:1, 63.6:1, and 69.6:1 for [(18)F]4, [(18)F]5, and [(18)F]6, respectively, at 2 h postinjection. Installation of aminohexanoic acid (AH) linkers in the phosphoramidate scaffold improved their PSMA binding and inhibition and was critical for achieving suitable in vivo imaging properties, positioning [(18)F]5 and [(18)F]6 as favorable candidates for future prostate cancer imaging clinical trials.


Asunto(s)
Amidas/farmacología , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Peptidomiméticos/farmacología , Ácidos Fosfóricos/farmacología , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico por imagen , Amidas/síntesis química , Amidas/química , Animales , Antígenos de Superficie , Relación Dosis-Respuesta a Droga , Radioisótopos de Flúor , Humanos , Masculino , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/diagnóstico por imagen , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Ácidos Fosfóricos/síntesis química , Ácidos Fosfóricos/química , Relación Estructura-Actividad , Células Tumorales Cultivadas
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